The present invention relates to liposome preparations capable of use in administration of organic solvent labile materials, such as whole live or attenuated cells, to human or animal bodies. Such preparations have utility in delivery of labile bioactive materials whereby a slow release is provided which may b e targeted to specific body areas. A method for the manufacture of such preparations is also provided.
The use of liposomes in the administration of vaccine agents is well known, and their adjuvant activity has been demonstrated by numerous studies into immunopotentiation of a large variety of bacterial, viral, protozoan, protein and peptide vaccines; see reviews by Gregoriandis G (1990) Immunol Today, 11, 89-97 and Alving C R (1991) J Immunol Meth, 140, p1-13.
These studies have all been carried out using liposomes produced by techniques which generate vesicles of submicron average diameter (see Gregoriadis G (ed) (1993) Liposome Technology, 2nd Edition, Volumes I-III CRC Press, Boca Raton, 1993) which are capable of accomodating peptides and proteins, but not capable of efficiently carrying larger vaccines. Such larger vaccines include a number of attenuated or killed viruses and bacteria such as measles, polio virus, Bordetella pertussis, Bacille Calmette-Guerin and Salmonella typhi (see Mimms C A et al (1993) Medical Microbiology, Chapter 36, Mosby).
Although most of these vaccines are highly immunogenic, there are circumstances where their administration in sufficiently large liposomes may be a preferred alternative. For instance, in the case of multiple vaccines consisting of a mixture of soluble and particulate (eg. microbial) antigens or vaccine formulations also containing cytokines, simultaneous presentation of all materials to immunocompetant cells via a common liposome carrier may be advantageous in terms of improving the immunogenicity to antigens.
Furthermore, liposomes incorporating antigenic material in their aqueous phase are known to prevent interaction of the antigen with its antibodies in pre-immunized animals and ensuing allergic reactions or antigen neutralisation (Gregoriadis and Allison (1974) FEBS Lett., 45, 71-74. It can thus be seen that liposomes could be beneficial if employed as carriers for administration of vaccines to infants for prophylaxis against agents for which maternal antibodies were present, eg, such as measles, or to individuals with hypersensitivity to vaccine contaminants.
It is known to incorporate particulate materials into large liposomes having average diameter up to 9.2 xcexcm by methods wherein solvents such as chloroform are formed into spherules containing smaller water droplets (see Kim and Martin (1981) Biochimica et Biophysica Acta, 646, 1-9). Using this technique materials such as Collagen, DNA and bacteria (Streptococcus salivarius) were entrapped, but it was noted that labile globular proteins such as serum albumen and haemoglobin did not allow liposome formation, presumably due to surface denaturation, and that protein denaturation occurred. Such method is unsuitable for the encapsulation of labile materials due to the damaging and cytotoxic effects of the organic solvent, and certainly unsuitable for the encapsulation of whole (live) or attenuated bacteria, protozoa, viruses or multicellular animal or plant cells.
Methods for entrapping soluble materials in liposomes without use of organic solvents in the encapsulation step have been known for several years (see Kirby and Gregoriadis (1984) Liposome Technology, Vol I, Gregoriadis G (ed), CRC Press, Inc Boca Raton, Fla., pp 19-28; Deamer and Uster (1983) Liposomes, Ostro M J (ed) Marcel Dekker, Inc, NY. pp27-51; Deamer and Barchfield (1982) J Mol Evol 18, 203-206), and are based upon a method which dehydrates preformed liposomes then rehydrates them in the presence of the soluble materials. In these methods the soluble materials enter with water as the liposomes fuse together resulting in material being entrapped in multilamella liposomes. The liposomes used were 40 to 80 nm in diameter before freeze drying and the multilamellar product vesicle volume resulting was still smaller. Such volume and structure are unsuitable for encapsulating micrometer size and/or living materials, and entrapment levels for soluble drugs are not as high as for unilamella liposomes due to relatively low surface area for entry into the vesicles. The same technique has also been applied to small unilammela liposomes for the purpose of encapsulating aqueous solutions (see EP 0171710).
The aforesaid process is relatively mild and has been used to successfully encapsulate labile solutes such as factor VIII (see Kirby and Gregoriadis (1984) Biotechnology, 2, 979-984) and tetanus toxoid (Gregoriadis et al (1987) Vaccine, Vol 5, p145-151). It relies upon solute entering the liposomes as they form while rehydration water enters. Despite such work on solutes, there has still not been provided a method for the encapsulation of whole (live) or attenuated organisms, cells or other insoluble structures bearing labile entities, without damaging them; whether bacterial, protozoan, viral or otherwise.
Furthermore, no method has yet been provided for encapsulating water labile soluble materials in larger liposomes, whether unilamellar or multilamella, that would allow targeting at specific tissues with still higher quantities of material.
The present inventors have now surprisingly found that dehydration/rehydration is capable of successful encapsulation of insoluble particulates such as whole live or attenuated organisms, cells, or microscopic water insoluble structures having organic solvent labile activity, whereby organisms are not killed and activity is retained. The invention allows micrometer sized unilamella and multilamella liposomes to be produced, (ie. 0.1-50 xcexcm diameter liposomes) which in contrast with the liposomes of the prior art, are capable of entrapping micrometer size and/or living material, and have inner vesicles of relatively high capacity, being similar in size to their outer diameter in the case of the unilamella giant liposomes.
It is particularly surprisingly that (i) when micrometer sized liposomes are dehydrated then rehydrated in this manner, unilamella liposome structure is retained which offers improved capacity for soluble material as well as the ability to retain particulates described above and (ii) where conditions are used such that multilamella liposomes are formed containing insoluble or undissolved material they are of micron size rather than the previously obtained 40 to 80 nm in diameter.
Thus in a first aspect of the invention there is provided a method for forming liposomes of greater than 0.1 xcexcm diameter, preferably greater than 1 xcexcm diameter, containing undissolved or insoluble particulate biologically, chemically or physically active material comprising (a) forming unilamellar liposomes (b) freeze drying the liposomes so formed and then (c) rehydrating them in intimate admixture with the undissolved or insoluble material to be contained therein.
Where unilamella liposomes are to be produced step (a) forms liposomes of greater than 0.1 xcexcm in diameter and uses these in step (b). Where multilamella liposomes are to be produced the size of the liposomes need not be fixed in step (a), but determined by the undissolved or insoluble material with which they are preferably freeze dried with in step (b) prior to rehydration in step (c).
The freeze drying step is, in the case of both unilamella and multilamella liposomes, preferably carried out on a mixture of the liposomes and material to be entrapped and may be carried out by known methods for freeze drying liposomes. The rehydration step is preferably controlled such that the number of liposomes destroyed by osmotic pressures induced by solute concentrations generated by water entering the vesicles is minimised.
In a second aspect the present invention further provides liposomes produced by the method of the first aspect of the invention, and particularly provides liposomes characterised in that they are over 0.1 xcexcm, preferably over 1 xcexcm, in diameter and contain biologically, chemically or physically active materials that would have their activity damaged or destroyed by contact with organic solvents.
It is particularly preferred that substantially all of any organic solvent used in the step of liposome preparation (a) is removed prior to the rehydration step (c), most conveniently before the freeze drying step (b).
Preferred particulate materials are microorganisms, including bacteria, protozoa and viruses, plant or animal cells or water insoluble structures having organic solvent labile biochemical or immunological activity. It should be noted however that any water insoluble particulate may be encapsulated using the method. For example catalysts or drugs that are sparingly soluble may also be so incorporated such that slow release into the a patients body may be achieved. However, as organic solvents would not be expected to adversely affect these materials such method would be merely an option that might be used in place of the known methods; the main advantage of this preferred aspect of the present method being realised in its application to the organic solvent sensitive microorganisms, cells and materials, and in yielding increased capacity with multilamella liposomes.
Step (a) of forming the liposomes may use any of the known methods, including those involving use of solvents in their manufacture, as these remove such solvents to leave hollow bodies; the hollows forming the vesicles into which the solutions, microorganisms, cells or insoluble structures are to be situated after entrapment. Typically, for unilamella liposome production, the step (a) will comprise a method for the formation of so called xe2x80x98giant liposomesxe2x80x99 of suitable size for encapsulating the material added in step (c); such method being suitably eg. that of Kim and Martin described above. Most preferably these will be of xe2x80x98micrometerxe2x80x99 or xe2x80x98micronxe2x80x99 sizexe2x80x99, ie. herein defined as from 0.1 xcexcm to 50 xcexcm in diameter, more preferably 1 xcexcm to 30 xcexcm. For multilamella liposome production standard dehydration/rehydration vesicles (DRVs) may be formed.
For most satisfactory encapsulation rates the step of freeze drying step (b) is carried out with the material to be encapsulated already intimately mixed with the liposomes. In this manner relatively high encapsulation rates have been achieved whereas when the mixture of liposomes and material for encapsulation is not intimate enough, little or no incorporation is more likely. This is not the acse where solutions are being incorporated as in the prior art.
Step (c) may be carried out by any rehydration method that allows the liposomes to admit the material to be encapsulated. Conveniently this is found to include a procedure wherein water in any readily available form, eg. distilled or tap water or a buffer solution, is added in a controlled manner to the freeze dried mixture of liposomes and material to be encapsulated. Preferably distilled water is first added in order to avoid still further osmotic stress to the liposome structure. Conveniently this is added in small quantity sufficient just to produce a suspension, followed after several minutes, preferably 20 to 40 minutes, eg. 30 minutes, by a similar amount of a buffer which is suitable for allowing the material to be encapsulated to retain its desired activity; one such suitable buffer being phosphate buffered saline (PBS) pH7.4. Again, the buffer is preferred at this stage in order to balance the high osomotic pressure of the solution forming in the vesicles of the liposomes as the materials present before the drying step are slowly rehydrated.
The suspension so obtained is preferably mixed with a larger volume of buffer, eg. PBS, after a further period, again preferably 20 to 40 minutes, preferably for about 30 minutes. The liposomes are typically freeze-dried from a suspension of liposomes, and the total volume of water and saline added in rehydration is conveniently sufficient to provide from 1 to 10 times that of the volume of the suspension, although no particular limits are placed here.
The rehydration step may be carried out at any temperature compatible with viability or retention of the desired activity of the material that is to be encapsulated. Thus typically any temperature from 0xc2x0 C. to 60xc2x0 C. might be selected where high melting point lipids are used in the liposomes and the material to be encapsulated is resistant to this temperature. Where living materials or proteins are used then 0xc2x0 C. to 40xc2x0 C. would be more usual, preferably 10xc2x0 C. to 30xc2x0 C. It will be realised however that certain organisms and proteins will be capable of treatment at much higher temperatures.
In order to maximise survival of the labile activity and the integrity of the liposomes in storage it may be advantageous to incorporate a cryoprotectant to counter the affects of freezing and water loss. This is preferably added after rehydration step (c) has been effected. Typical of such protectants are sugars and their derivatives, particularly sugars such as trehalose (see Crowe and Crowe in Liposome Technology (1993) V Vol I, pp229-249, CRC Press Inc, Boca Raton), with techniques for using this being well known to those skilled in the art.
The composition of the preformed liposomes provided in step (a) is also not particularly limited, but must allow for stable formation of liposomes having sufficient capacity to hold the material to be encapsulated. Typical lipid compositions used for formation of so called xe2x80x98giant liposomesxe2x80x99 and DRVs comprise phosphatidylcholine (PC) or distearoylphosphatidyl choline (DSPC), and these are optionally supplemented with components such as cholesterol, phosphatidyl glycerol (PG) and/or triolein (TO). Other components known in the art or developments thereof which provide liposome stability or induce vesicle formation may also be used.
Formation of giant liposomes from such mixtures is conveniently achieved by mixing a chloroform solution of these components with a sucrose solution to form and emulsion, then mixing that with a similar ether water emulsion to provide a water-in -oil-in-water emulsion, from which are removed the organic solvents to generate liposomes. Formation of DRVs may conveniently be achieved by dissolving equimolar PC, or DSPC, and cholesterol in chloroform and rotary evaporating the mixture to leave a thin film of lipid on a flask wall. This film is then disrupted at 4xc2x0 C. (for PC) or 60xc2x0 C. (for DSPC) with 2 ml distilled water followed by probe sonication for 2 minutes to yield small unilamella vesicles (SUVs). This suspension is then suitable for freeze drying with material to be encapsulated whereby the multilamella DRVs of greater than 0.1 xcexcm diameter form.
In a third aspect of the present invention there is provided a method for separation of liposomes of the invention from non-entrapped microorganisms, cells or water insoluble structures characterised in that it places a mixture of the two on a density gradient and centrifuges it, the fractions of the gradient are removed, those containing the separated liposomes collected, and the liposomes separated from these by conventional methods; the free materials usually being collected in the lower fractions and the liposomes in the upper fractions. Preferably the gradient is a 0.4M to 4M sucrose gradient or gradient including an equivalent density range or analogous sugar. Where separation from soluble materials is also required the liposomes are centrifuged at approximately 600xc3x97g in buffer, eg. PBS, whereby they are collected as a pellet.
A fourth aspect of the present invention is therefor provided in the form of liposomes of the invention free from non-entrapped form of the undissolved or insoluble particulates they contain. Such forms are of course advantageous determination of dosage given.
As stated in the introductory paragraphs above, it is sometimes advantageous to present more than one agent to a target area of a patient simultaneously, and the present invention provides such advantage wherein the liposomes of the invention, the method of preparing them and the method of separating them from non-entrapped materials all incorporate or cater for handling of water soluble agent. Thus the liposomes produced by the method of the second aspect of the invention may contain living or attenuated microorganisms, cells and/or water insoluble structures together with water soluble agents such as vaccines, antibodies, antigens or enzymes.
Thus this method of preparing the liposomes of the invention will, when soluble materials are also to be incorporated, include the soluble material with the insoluble material with the liposomes in the rehydration step, preferably in the freeze drying step, and the method for separating non-entrapped material from the liposomes will utilise both the density gradient and buffer centrifugation methods.
A further aspect of the present invention, by virtue of the aforesaid aspects unique advantages, provides novel liposomes characterised in that they contain whole live or attenuated microorganisms, plant or animal cells, or water insoluble non-living structures having organic solvent labile biochemical or immunological activity. The latter will include killed organisms that retain a desired activity that is labile to organic solvent treatment. The liposomes of the present invention can readily be identified in that their content can be released and demonstrated to have retained the ability to be cultured and/or to illicit biochemical or immunological responses. In addition to bacteria, protozoans, cells or viruses, the liposomes of the present invention may comprise inanimate structures such as cytokine, enzyme, antigen or antibody bearing support materials, such as latex beads or other polymeric support bodies.
In a preferred form of this aspect the liposomes, and thus the approximate size of their vesicles, are from 0.1 xcexcm to 50 xcexcm in diameter, preferably from 1 xcexcm to 30 xcexcm, and conveniently 1 xcexcm to 14 xcexcm, with a convenient mean diameter being 5.5 xcexcm+2.2; but vesicle size required will necessarily be dictated by the amount or size of solution, microorganism, cell or water insoluble structure that is intended to be encapsulated. To this end the means by which the liposomes are initially formed is not important, and thus variation in vesicle size is potentially unlimited as a method is provided for incorporating the labile materials, particularly microorganisms or insoluble structures, into already formed liposomes without killing or inactivating them or destroying liposome integrity.
Use of the liposomes of the present invention allows targeting of the macrophages, phagocytes and/or antibody producing cells of the body specifically, by virtue of the fact that the preferred liposomes, as stabilised with cholesterol, PG or equivalent materials, do not substantially release their particulate content spontaneously. Thus the fate of the particulate material tends to be in processing by macrophages or phagocytes whereby the immune response and related effects, eg, of cytokines, are enhanced. Furthermore, the fact that the particulate is protected from circulating antibodies by the lipid, until such encounter with the macrophages or phagocytes, ensures maximal presentation to the immune system and antibody producing cells.
The liposomes and methods of the present invention will now be illustrated by reference to the following Figures, non-limiting Examples, and Comparative Example. Many other suitable liposomes and methods for their preparation falling within the scope of the invention being readily evident to those skilled in the art in the light of these.
Formation of Giant Unilamella Liposomes
In these examples the giant liposomes are preformed, have a mean diameter of 5.5xc2x10.2 xcexcm and are mixed with the particulate or soluble materials and subsequently subjected to controlled rehydration.
Generated liposomes were found to maintain their original mean diameter and diameter range and to contain up to 26.7% (mean value) of the added materials. Particulate-containing liposomes could be freeze-dried in the presence of trehalose with most (up to 87%) of the entrapped material recovered within the vesicles formed on reconstitution with saline.
The sources and grades of egg phosphatidylcholine (PC), distearoyl phosphatidylcholine (DSPC), cholesterol, immunopurified tetanus toxoid and trehalose have been described elsewhere (Davis and Gregoriadis, 1987, Immunology, 61, 229-234). Phosphatidyl glycerol (PG) and triolein (TO) were from Lipid Products (Nuthill, Surrey) and Sigma Chemical Company (London) respectively. Killed Bacillus subtilis (B.subtilis) and Bacille Calmette-Guerin (BCG) were gifts from Dr Bruce Jones (Public Health Laboratories Service, Porton Down, Salisbury, Wilts) and Dr J. L. Stanford (Dept of Medical Microbiology, UCL Medical School, London) respectively. Radiolabelling of tetanus toxoid, B.subtilis, and BCG with 125I was carried out as described previously (Kirby and Gregoriadis, 1984 as above). Labelling of B.subtilis with fluorescein isothiocyanate (FITC) (Sigma) was carried out by incubating the bacteria in 1 ml 0.1M sodium carbonate buffer (pH9.0) containing 1mg FITC for 24 h at 4xc2x0 C/ (Mann and Fish,(1972) Meth. Enzymology, 26, 28-42). All other reagents were of analytical grade.